The Development of a Rectal Enema As Microbicide (DREAM) Program addresses the critical need to develop a highly effective, safe, and acceptable microbicide enema with the promise of greater adherence as a more behaviorally-congruent alternative for the prevention of rectal HIV infection. The overall goal of the program is to develop a single dose rectal enema to deliver a tenofovir (TFV) prodrug capable of providing one week of protection. This strategy builds upon proven high levels of efficacy of TFV-based pre-exposure prophylaxis (PrEP) in adherent persons and directly targets the greatest weakness of PrEP regimens - prophylactic failure due to poor adherence. Given the common practice of rectal douching with an enema prior to receptive anal sex, our PrEP delivery via enema directly addresses adherence problems by requiring little or no behavioral change compared to other oral and topical approaches. Topical delivery also significantly reduces systemic exposure compared to oral and injectable approaches. Through pharmacokinetic enhancements to increase TFV bioavailability and provide sustained release we will study three TFV prodrugs with far greater tissue and cellular uptake than TFV itself - TFV disoproxil fumarate, TFV alafenamide fumarate, and CMX157. Integrated with 3 other projects and 3 cores in the DREAM Program, Project 4 focuses on preparation and execution of a pre-phase I clinical study to compare a single dose (1) optimized TFV enema, (2) best TFV prodrug enema, and (3) TFV 1% gel in a cross-over design to decide whether and which TFV enema to advance to clinical development. We propose the following specific aims: Aim 1: Prepare an IND for clinical study of TFV prodrug enema. Aim 2: Perform pharmaceutical analysis and product compounding to support the TFV prodrug enema IND and provide supporting materials for dose preparation at clinical sites. Aim 3: Perform pre-clinical toxicology testing to support TFV prodrug enema IND Aim 4: Conduct an exploratory, double-blinded, randomized, pharmacokinetic-pharmacodynamic, acceptability, and safety study of a TFV enema, TFV prodrug enema, and TFV 1% gel (CLIN 03).